The beneficial effects of the long-chain polyunsaturated fatty acids (PUFA) that are characteristic of marine lipids, especially cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on lowering serum triglycerides are now well established. These compounds are also known for other cardioprotective benefits and other biological effects. Among the most frequently mentioned benefits are those related to the prevention of and the treatment of inflammation, neurogenerative diseases, and cognitive development abnormalities. The public is becoming increasingly aware of the health benefits of fish oil and DHA and EPA concentrates, as it is evidenced from global sales of polyunsaturated fatty acids (PUFA). For instance, the sales of PUFA rose by 50% in 2002, and they were the only ones out of all categories of nutraceuticals that made a significant increase in sales.
Several methods of producing PUFA concentrates from marine oils are known, for example, selective lipase hydrolysis, PUFA complexation using urea (or more sophisticated molecular guest-host frameworks involving metric control), and a physical removal of unwanted components by fractionation. U.S. Publication No. 2004/0236128 describes the separation of EPA from DHA by precipitating EPA magnesium salt.
Fractionation involving molecular distillation is usually conducted on ethyl esters prepared from the starting triglycerides since they are more volatile than corresponding triglycerides. However, there is a controversy as to whether ethyl esters of PUFA are as bioavailable as their triglyceride counterparts. Therefore, there is a need to re-esterify the esters to the corresponding triglycerides.
The reaction describing the formation of triglycerides from the ethyl esters of fish oil is transesterification. Transesterification is a process where an ester is converted into another ester through interchange of the alkoxy moieties.

The reaction is an equilibrium process and the transformation occurs essentially by simply mixing the two components. However, it has been shown that the reaction is accelerated by Lewis acid catalysts (such as BBr3, AlCl3 etc., embedded in polystyrene-divinylbenzene), Bronsted acid catalysts such as HCl, H3PO4, H2SO4, p-TosOH, or basic catalysts such as metal alkoxides, metal hydroxides, and metal carbonates.
It has been apparent, however, that the transesterification reaction under these conditions does not meet in many cases the requirements of modern synthetic chemistry, i.e., highly efficient and regioselective reaction conditions. Thus efforts have continued to conduct transesterifications under milder conditions and to control randomization. Among the chemical catalysts developed, distanoxane was found to be effective for transesterification of various types of esters; however, this catalyst is difficult to prepare and is not commercially available, at least not in large quantities. The titanate-mediated transesterification method is extremely mild but fails to achieve certain kinds of transesterifications. Similarly, DMAP catalyzed reactions or reactions in the presence of tin-based superacids have different profiles of reaction selectivity. Successful attempts have also been made to perform transesterifications using zeolites, neutral chromatographic alumina, or kaolinites.
In spite of the advances in modern synthetic chemistry, the most popular industrial catalysts are strong bases. They are inexpensive, but because of their nature they generate a significant degree of side products, particularly at high temperatures necessary to achieve the desirable yields. In addition, they are not regioselective and lead to side reactions. Although they may be good choices for stable and structurally suitable chemical entities, they are not preferred for complex products such as, for example, fish oils. In the case of fish oils, enzymatically catalyzed transesterifications are a viable alternative, since they are regioselective and generate virtually no side products. However, their drawback is generally their cost.
Therefore, there is a need to transesterify complex esters such as, for example, polyunsaturated fatty acid esters to more useful esters. There is also a need for the efficient esterification of complex carboxylic acids such as fatty acids. The procedures should have a high efficiency with respect to transesterification and esterification and yet not produce undesirable side-products. The process should also be relatively inexpensive for commercial applications. Finally, the esters produced by the methods described herein should be ready to be incorporated into numerous food products without the requirement of removing additional impurities from the transesterification process. The immobilized enzymes and methods of use described herein satisfy these long-felt needs.